Functionalized environmentally benign nanoparticles

ABSTRACT

This invention is directed to the preparation and applications of internally and/or externally functionalized environmentally benign nanoparticles (EbNPs), which are produced by a three step procedure: (1) synthesis of native EbNPs, (2) functionalization with active agents, and (3) additional surface property customization via one or more modifier(s).

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. 554,871awarded by the U.S. Environmental Protection Agency. The government hascertain rights in the invention.

1. FIELD OF THE INVENTION

This invention relates generally to the preparation and applications ofinternally and/or externally functionalized environmentally benignnanoparticles (EbNPs), which are produced by a three-step procedure: (1)synthesis of native EbNPs, (2) functionalization with active agents, and(3) additional surface property customization via one or moremodifier(s).

2. BACKGROUND OF THE INVENTION 2.1. Introduction

Engineered nanoparticles exhibit unique and useful physical, chemical,and biological particle-specific attributes that may help to solvepressing challenges of mankind in industries including life sciences,energy, and health care. However, nanoparticle waste has been recognizedas a potential health hazard,¹ as the post-utilization activity ofengineered nanoparticles combined with their persistence may result inshort and long-term toxicity in humans and the environment.² Forexample, it has been found that the physical and chemicalcharacteristics of persistent nanoparticles (PNPs), and therefore, theiractivity, may not change even after high-temperature treatments insolid-waste incineration plants.³ One way to minimize thepost-utilization hazard of nanoparticles is to minimize their residencetime and presence in the environment. Employing degradable nanoparticleswith matching functionality to PNPs may serve as a suitable solution.

Lignin, the most abundant aromatic polymer in nature,⁴ has an amorphousstructure and is biodegradable. Lignin covalently crosslinks the cellwalls of plants, and plays a vital role in plant health, growth, anddevelopment.⁵ When extracted from biomass, the structure of modifiedlignin varies depending on the initial plant source and the method ofisolation. Lignin obtained via the organosolv process, such as HighPurity Lignin (HPL), is strongly hydrophobic, does not incorporate anysulfur containing groups, and therefore, preserves best the structure ofnative lignin of all processed lignins.⁶ However, the most commonextraction method is the Kraft pulping processes.⁷ INDULIN AT lignin(IAT), a modified lignin that contains a small number of hydrophilicthiol groups, is recovered by this process. In aqueous systems, matrixesof IAT have shown high absorbance capabilities of heavy metal ions forenvironmental remediation purposes.^(8, 9) Cationic metal ions areelectrostatically attracted to IAT, which is negatively charged inaqueous solution due to deprotonation of its main functional groups(Table 6). Recently, the synthesis of pH-stable IAT-basedenvironmentally benign nanoparticles (EbNPs) in ethylene glycol wasreported.¹⁰ Hence, we propose that by infusing IAT EbNPs with functionalmetal ions, it will be possible to synthesize degradable nanoparticlesthat match the nanoparticle functionality of the respective metal PNPs,while increasing post-utilization safety.

Silver nanoparticles (AgNPs) are among the most widely employed PNPs, astheir broad-spectrum antimicrobial properties allow them to combatbacteria strains exhibiting antibiotic resistance,¹¹ which are reportedin human pathogens including Escherichia coli (E. coli)¹² andPseudomonas aeruginosa (P. aeruginosa).¹³ As infection control measurescan minimize the spread of drug-resistant bacteria,¹⁴ and therefore thepotential for nosocomial infections,¹⁵ silver-containing products mayfind increasing utilization in the medical sector to prevent bacteriagrowth on catheters,¹⁶ prostheses,¹⁷ and dental materials,¹⁸ and toreduce the infection potential of burn wounds.¹⁹ In addition, with theemergence of antimicrobial PNPs in textiles,²⁰ water filters,²¹ andother consumer products, the human exposure potential to PNPs with theirassociated risks increases. Human skin exposure studies indicate thatAgNPs can be released from antibacterial fabrics into liquids resembling“sweat”.²² Studies on commercially available wound dressings proved thatdressings containing AgNPs exhibit stronger cytotoxic effects towardkeratinocytes than do PNP-free counterparts.²³ In vitro studies onmammalian fibroblasts have revealed that AgNP can induce apoptosis. Inthis context, AgNP may potentially affect human health.^(24, 25, 26)

Several methods for the preparation of antimicrobial silver-basednanoparticle systems have been reported. Most procedures employ highlyreactive reducing agents such as sodium borohydride (NaBH₄) or hydrazine(N₂H₄) to reduce silver ions to metallic silver. Green synthesis methodsof producing AgNP can reduce the environmental impact during fabricationgiven that no harsh solvents or reducing agents are employed.^(27, 28)However, due to the persistent nature of AgNPs, the problem ofpost-utilization toxicity associated with non-degradable nanoparticlesremains unaddressed.

3. SUMMARY OF THE INVENTION

In particular non-limiting embodiments, the present invention provides:

-   1. A nanoparticle comprising:    -   a biodegradable biopolymer core, an antiviral or cytotoxic agent        loaded on the biodegradable core, and a cationic layer coating        the biodegradable core with the bioactive agent.-   2. The nanoparticle of claim 1, wherein the biodegradable biopolymer    core is either a lignin, a modified lignin, or a linear, branched,    or cross-linked polysaccharide.-   3. The nanoparticle of claim 1, wherein the biodegradable biopolymer    core is a plant- or animal-derived biopolymer.-   4. The nanoparticle of claim 3, wherein the plant- or animal-derived    biopolymer core is a cellulose, a chitin, a chitosan, a    hemicellulose, a lignocellulose, a modified cellulose, a modified    chitosan, a modified lignin, a protein, or a combination thereof.-   5. The nanoparticle of claim 4, wherein the chitosan is a medium or    high molecular weight chitosan or a derivative thereof.-   6. The nanoparticle of claim 4, wherein the modified cellulose is    cellulose acetate, cellulose nitrate, cellulose propionate, ethyl    cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose    phthalate, hydroxypropyl methylcellulose acetate succinate, or    methyl cellulose, or a derivative thereof.-   7. The nanoparticle of claim 4, wherein the modified chitosan is a    low molecular weight chitosan, a chitosan with amino groups in the    backbone, or a derivative thereof.-   8. The nanoparticle of claim 4, wherein the modified lignin is    sulfonated or unsulfonated lignin.-   9. The nanoparticle of claim 1, wherein the antiviral or cytotoxic    agent is an antiviral agent.-   10. The nanoparticle of claim 1, wherein the antiviral or cytotoxic    agent is a biocide, a cationic metal, a catalyst, a fumigant, a    herbicide, a pesticide, a photocatalyst, or a semiconductor.-   11. The nanoparticle of claim 10, wherein the biocide is an    algaecide, a bactericide, or a fungicide.-   12. The nanoparticle of claim 11, wherein the fungicide is captan,    chlorothalonil, cyrodinil, folpet, mepanipyrim, pyrimethanil,    sulfur, or vinclozolin.-   13, The nanoparticle of claim 11, wherein the fungicide is an    ethylenebisdithiocarbamate or a natural fungicide.-   14. The nanoparticle of claim 13, wherein the    ethylenebisdithiocarbamate is mancozeb, maneb, metiram, nabam, or    zineb.-   15. The nanoparticle of claim 13, wherein the natural fungicide is    ampelomuces quisqualis, cinnamaldehyde, cinnamon essential oil,    jojoba oil, monocerin, neem oil, rosemary oil, or tee tree oil.-   16. The nanoparticle of claim 10, wherein the cationic metal is Ag⁺,    Ag²⁺, Ag³⁺, Co²⁺, Cu²⁺, Fe²⁺, Ni²⁺, or Zn²⁺.-   17. The nanoparticle of claim 10, wherein the fumigant is    1,3-dichloropropene, chloropicrin, formaldehyde, iodoform, metam    sodium, methyl bromide, methyl iodide, methyl isocyanate, phosphine,    or sulfuryl fluoride.-   18. The nanoparticle of claim 10, wherein the herbicide glyphosate,    triclopyr, 1,1′-dimethyl-4,4′-bipyridinium ion (paraquat) or a    chemical derivative, analogue or salt thereof.-   19. The nanoparticle of claim 10, wherein the herbicide is a    chloracetanilide herbicide, glyphosate herbicide, an imidazolinone    herbicide, an organic herbicide, a phenoxy herbicide, a phenylurea    herbicide, a triazine herbicide, or a triazolopyrimidine herbicide.-   20. The nanoparticle of claim 19, wherein the chloracetanilide    herbicide is acetochlor, alachlor, butachlor, metolachlor, or    propachlor.-   21. The nanoparticle of claim 19, wherein the imidazolinone    herbicide is imazapyr, imazamethabenz-methyl, imazapic, imazethapyr,    imazamox or imazaquin.-   22. The nanoparticle of claim 19, wherein the organic herbicide is    corn gluten meal, vinegar, D-limonene, or monocerin.-   23. The nanoparticle of claim 19, wherein the phenoxy herbicide is    2,4-Dichlorophenoxyacetic acid, 2,4,5-Trichlorophenoxyacetic acid,    2-Methyl-4-chlorophenoxyacetic acid,    2-(2-Methyl-4-chlorophenoxy)propionic acids,    2-(2,4-Dichlorophenoxy)propionic acid, or    2,4-Dichlorophenoxy)butyric acid.-   24. The nanoparticle of claim 19, wherein the phenylurea herbicide    is N′-(3,4-dichlorophenyl)-N,N-dimethylurea (diuron),    1,1-dimethyl-3-[3-(trifluoromethyl)phenyl (fluometuron), or    N,N-dimethyl-N′-[4-(1-methylethyl)phenyl (isoproturon).-   25, The nanoparticle of claim 19, wherein the triazine herbicide is    ametryn, atrazine, cyanazine, prometon, prometryn, propazine,    simazine, terbuthylazine, or terbutryn.-   26. The nanoparticle of claim 19, wherein the triazolopyrimidine    herbicide is clorasulam-methyl, diclosulam, florasulam, flumetsulam,    metosulam, penoxsulama, or pyroxsulama.-   27. The nanoparticle of claim 10, wherein the pesticide is an    avicide, an insecticide, a miticide, a molluscicide, a nematicide,    or a rodenticide.-   28. The nanoparticle of claim 27, wherein the insecticide is a    carbamate or a pyrethroid insecticide.-   29. The nanoparticle of claim 28, wherein the carbamate insecticide    is aldicarb, carbaryl, carbofuran, formentanate, methiocarb, oxamyl,    pirimicarb, propoxur, or thiodicarb.-   30. The nanoparticle of claim 28, wherein the pyrethroid insecticide    is allethrin, bifenthrin, cyhalothrin, lambda-cyhalothrin,    cypermethrin, cyfluthrin, deltamethrin, etofenprox, fenvalerate,    permethrin, phenothrin, prallethrin, pesmethrin, tetramethrin,    tralomethrin, or transfluthrin.-   31. The nanoparticle of claim 27, wherein the rodenticide is an    anticoagulants, brodifacouma, bromadiolonea, chlorophacinone,    difethialone, diphacinone, pindone, warfarin, nonanticoagulant,    bromethalin, cholecalciferol, strychnine, or zinc phosphide-   32. The nanoparticle of claim 10, wherein the semiconductor is Ag₂S,    CdS, CdSe, CdTe, Cu₂S, CuCl, CuO, Fe₂O₃, Fe₂S, Fe₃O₄, FeO, NiO,    TiO₂, ZnO, ZnS, ZnSe, or ZnTe.-   33. The nanoparticle of claim 1, wherein the bioadhesive layer is a    cationic polymer.-   34. The nanoparticle of claim 33, wherein the cationic polymer is a    polyamino-polymer.-   35. The nanoparticle of claim 34, wherein the polyamino-polymer is    branched polyethyleneimine (BPEI), polyallylamine hydrochloride    (PAH), polydiallyldimethylammonium chloride (PDADMAC),    polyethoxylated tallow amine (POEA), polyethyleneimine (PEI), or    polylysine.-   36. The nanoparticle of claim 1, wherein the bioadhesive layer    comprises primary, secondary, tertiary, or quaternized amines.-   37. The nanoparticle in claim 1, where the bioadhesive layer    comprises carbohydrates, polypeptides, lectins, proteins, or    antibodies or other molecules or materials with affinity to    microbes, viruses or seeds.-   38. The nanoparticle in claim 1, where the bioadhesive layer    comprises nanohairs, nanolatches-   39. The nanoparticle of claim 1, wherein the nanoparticle has a    diameter of about 10 nm to about 500 nm.-   40. The nanoparticle of claim 39, wherein the nanoparticle has a    diameter of about 20 nm to about 100 nm.-   41. The nanoparticle of claim 40, wherein the nanoparticle has a    diameter of about 50 nm to about 80 nm.-   42. A coated article comprising a surface wherein at least a portion    of the surface is coated with the nanoparticle of claim 1.-   43. The coated article of claim 42, wherein the coated article is an    air filter, an article of clothing, an article of hygiene, a    building material, a face mask, a food stuff package, a medical    device, or a seed.-   44. The coated article of claim 43, wherein the medical device is    bandage, a biological implant, a dressing, a medical scaffold, a    surgical instrument, or a wound covering.-   45. The use of the nanoparticle of claim 1 to impart antiviral or    cytotoxic properties to a substrate.-   46. A method for fabricating a nanoparticle, the method comprising:    contacting a solvent containing a dissolved biodegradable biopolymer    with an anti-solvent so as to form a biodegradable biopolymer core;    loading an antiviral or cytotoxic agent on the biopolymer core; and    coating the biopolymer core and the antiviral or cytotoxic agent    with a bioadhesive layer.-   47. A method for fabricating a nanoparticle, the method comprising:    altering the pH of a suitable solvent containing a dissolved    biodegradable biopolymer so as to form a biodegradable biopolymer    core;    loading an antiviral or cytotoxic agent on the biopolymer core; and    coating the biopolymer core and the antiviral or cytotoxic agent    with a bioadhesive layer.-   48. A method for fabricating a nanoparticle, the method comprising:    contacting an organic solvent containing a dissolved biodegradable    biopolymer with an aqueous solvent under suitable pH conditions so    as to form a biodegradable biopolymer core;    loading an antiviral or cytotoxic agent on the biopolymer core; and    coating the biopolymer core and the antiviral or cytotoxic agent    with a bioadhesive layer.-   49. A method for fabricating a nanoparticle, the method comprising:    contacting an aqueous containing a dissolved biodegradable    biopolymer with a polyelectrolyte under suitable conditions so as to    form a biodegradable biopolymer core;    loading an antiviral or cytotoxic agent on the biopolymer core; and    coating the biopolymer core and the antiviral or cytotoxic agent    with a bioadhesive layer.-   50. A nanoparticle fabricated according to the method of claim 46.-   51. A nanoparticle fabricated according to the method of claim 47.-   52. A nanoparticle fabricated according to the method of claim 48.-   53. A nanoparticle fabricated according to the method of claim 49.-   54. The nanoparticle of claim 4, wherein the protein is a prolamin    or a gluten-   55. The nanoparticle of claim 54, wherein the prolamin is gliadin,    hordein, secalin, zein, kafirin or avenin.-   56. The nanoparticle of claim 54, wherein the gluten is gladin or    glutenin.-   57. The nanoparticle of claim 1, wherein the biodegradable    biopolymer core is a byproduct of lignin degradation.-   58. The nanoparticle of claim 57, wherein the byproduct of lignin    degradation is humic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of the concept for making and using environmentallybenign bactericidal nanoparticles (EbNPs) compared to the present use ofAgNPs.

FIG. 2: Schematics of the concept for synthesizing HPL EbNP via thesolvent-antisolvent method.

FIG. 3: HPL EbNP synthesis. a) size as a function of initial HPLconcentration in solvent. b) TEM micrographs of native HPL EbNP.

FIG. 4: HPL EbNP size as a function of antisolvent dilution rate.

FIG. 5: HPL EbNP size and ξ-potential as a function of final pH.

FIG. 6: Schematics of the IAT EbNP synthesis methods.¹⁰

FIG. 7: Results of IAT EbNP synthesis via pH drop as a function of acidadded.

FIG. 8: IAT EbNPs size and ξ-potential vs. amount of HNO₃ added to 5 mlIAT0.5 wt % in ethylene glycol.

FIG. 9: IAT EbNP size and ξ-potential vs. pH.

FIG. 10: IAT UV-Vis calibration curve for UV-Vis IAT EbNP solvationmeasurement.

FIG. 11: IAT EbNP dissolution as a function of pH, obtained by UV-Vismeasurements.

FIG. 12: IAT EbNP stability as a function of ionic strength.

FIG. 13: DLVO modeling of IAT EbNP system. The interaction energy in kTis modeled as a function of particle separation distance in nm for threedifferent ionic strengths.

FIG. 14: Infusion of native IAT EbNPs. a, TEM micrograph of native IATEbNP. b, Schematics showing native IAT EbNP infusion with Ag⁺ in aqueoussolution. c, TEM image of silver-ion infused IAT EbNP.

FIG. 15: Ag⁺ calibration curve. Silver ion mV reading vs. Ag⁺ ppmloading.

FIG. 16: Determination of Γ_(max) and k for Langmuir adsorptionisotherm.

FIG. 17: Langmuir adsorption isotherm fit. Ag⁺ adsorption on IAT EbNPnanoparticles normalized on m² particle surface area vs. initial Ag⁺loading per m² particle surface area.

FIG. 18: EbNP diameter and ξ-potential vs, initial PDADMAC wt %.

FIG. 19: EbNP diameter and ξ-potential vs. initial PAH wt %.

FIG. 20: CFU on reference plate without agent (left) and CFU on testplate with antimicrobial agent (right).

FIG. 21: Wet method schematic for antimicrobial testing. (1) placementof active agent into centrifugal tubes, (2) addition of PBS buffer, (3)addition of bacteria solution, vortexing for 1 minute and 30 minutes,platting after 1 minute and 30 minutes, incubation of samples, andinvestigation of CFUs.

FIG. 22: Qualitative E. coli test—CFU reduction efficiency of selectedIAT EbNP, BPEI AgNP, and AgNO₃ samples. a, after 1 minute incubationtime. b, after 30 minutes incubation time.

FIG. 23: Quantitative Pseudomonas aeruginosa test—CFU reductionefficiency of selected BPEI AgNP, AgNO₃, PDADMAC, and IAT EbNP samples.a, after 1 minute incubation time. b, after 30 minutes incubation time.

FIG. 24: Schematics of the concept for making and using environmentallybenign bactericidal nanoparticles (EbNPs) compared to the present use ofAgNPs.

FIG. 25: Schematic of the concept for making and using functionalizedEbNPs. a, Repeating units of material building blocks. b, Synthesissteps for making functionalized EbNPs. c, Life cycle ofEbNPs—application and post-utilization activity.

FIG. 26: Qualitative E. coli test—CFU reduction efficiency of selectedIAT EbNP, BPEI AgNP, and AgNO₃ samples. a, after 1 minute contact time.b, after 30 minutes contact time.

FIG. 27: Quantitative Pseudomonas aeruginosa test—CFU reductionefficiency of selected IAT EbNP, BPEI AgNP, and AgNO₃ samples. a, after1 minute contact time. b, after 30 minutes contact time.

FIG. 28: Schematic of the hypothesis of the antimicrobial mechanism off-EbNPs-PDADMAC. a, Ag-EbNP-PDADMAC are electrostatically attracted tobacteria cell and b, can deliver the silver ions leading to cell death.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1. Concept Section 1

The schematic in FIG. 1 illustrates the three steps involved ingenerating sustainable antimicrobial nanoparticles, and their life cyclein comparison to that of persistent AgNPs. In one, non-limitingembodiment, the silver ion infused lignin-based EbNPs with positivesurface charge consist of: (1) a biodegradable EbNP core (negativelycharged IAT EbNPs); (2) an active agent (antimicrobial silver ionsadsorb on the negatively charged EbNP core); and (3) a surface modifier(polydiallyldimethylammonium chloride [PDADMAC], a positively chargedpolyelectrolyte). Both systems can attach to negatively charged bacteriacells. Both systems can release silver ions, which perform the desiredantimicrobial function leading to bacteria cell death. When examiningthe silver release from the AgNP system, first metallic silver has todissolve before it can be released in its ionic form into the bacteria.As the change of state from metallic to ionic silver may limit the rateat which silver ions are transferred from the AgNP system to the cell,this may reduce, overall, the antimicrobial efficiency of the system. Incontrast to the metallic silver in AgNPs, ionic silver is alreadyavailable in Ag-EbNPs-PDADMAC at contact with the cell. Therefore,Ag-EbNPs-PDADMAC may be capable of releasing silver ions more readily.This may result in high antimicrobial efficiency and rapid Ag depletionof the Ag-EbNPs-PDADMAC system. At the end of the lifecycle, bothsystems may eventually be released into the environment as nanomaterialwaste. Moreover, as AgNPs may stay active, releasing reactive silverions post-utilization, they represent persistent nanoparticle waste thatcould result in hazards for humans and the environment. On the otherhand, the Ag-EbNPs-PDADMAC system, which is depleted of silver ions, isrendered inactive and will degrade over time; hence, Ag-EbNPs-PDADMACmay increase post-utilization safety for humans and the environment.

Here, we report non-limiting, exemplary data on the synthesis of nativeHPL and IAT EbNPs, the infusion of native IAT EbNPs with silver ions,the surface charge modification of the system with PDADMAC, and thequantification of antimicrobial efficiencies for opportunistic humanpathogens E. coli and P. aeruginosa. In addition, we provide ahypothesis to explain the antimicrobial mechanism associated withAg-EbNPs-PDADMAC.

1. RESULTS 1.1 Synthesis and Characterization of HPL EbNPs

We synthesized native HPL EbNPs via the solvent-antisolventprecipitation method. As illustrated in FIG. 2, hydrophobic HPL is firstdissolved in a good solvent, which is acetone, and rapidly transferredinto an antisolvent, which is Millipore H₂O. Upon change of media, HPLmay precipitate out as stable negatively charged EbNPs. The mainparameters controlling the size and size distribution of these EbNPsinclude the initial HPL loading in the solvent, and the rate of dilutionwith antisolvent.

The data on the effect of the initial HPL loading in the solvent on thefinal EbNP size are shown in FIG. 3. The TEM images may indicatespherical particles with diameters below 100 nm. The preparation of thesamples included the following two steps (1) 1 ml of HPL dissolved inacetone was placed in a 20 ml scintillation vial, (2) 9.21 ml ofMillipore H₂O was rapidly added to the solution immediatelyprecipitating out HPL EbNPs. The samples obtained were then furtherdiluted to 0.05 wt %, which is a suitable concentration for sizemeasurements with dynamic light scattering (DLS). The EbNP z-averagediameters increased with increasing amount of HPL in the initialsolvent. Repeated testing, as indicated with error bars, shows that thetarget diameters are reproducible. While the polydispersity widthincreases with particle size, the polydispersity indexes, whichfluctuate mainly between 0.05 and 0.20, did not show a distinct trend.The wide polydispersity width observed could be a consequence of thebroad molecular weight size distribution of the raw material HPL. Thestability of the particle suspensions over time was confirmed with asize measurement performed after 84 days, which did not show anysignificant change in diameter.

1.1.1 HPL EbNP Dilution Rate Study

We investigated the effect of the stock solution dilution rate on theHPL EbNP size. As reported in FIG. 4, the particle size decreases withincreasing dilution rate. Up to a dilution rate of 218 ml/minute, therate was adjusted with a syringe pump. For the dilution rates that couldnot be achieved with a syringe pump, the ones at 420 ml/minute and 1100ml/minute, we utilized a 10 ml hand pipette and performed a videoanalysis on the pipette tip to calculate the actual dilution rate.Overall, we developed a facile method to synthesize pH-stable HPL EbNPswith size control in the range of 50 to 250 nm in diameter.

1.1.2 HPL EbNP pH Stability

The stability of HPL EbNPs against pH change was tested in dialyzed 0.10wt % HPL EbNP samples, which were diluted down to 0.05 wt %. To adjustthe pH of the EbNP suspensions, callibrated amounts of HNO₃ or NaOHsolution were added. As indicated in FIG. 5, the pH stability of theEbNPs ranges from 3.25 to 10. Below a pH value of 3.25, the EbNPs showsigns of instability and eventually aggregate as indicated with thethreefold size increase at pH 2.91, which is accompanied with anapparent ξ-potential drop. When investigating the samples above pH 10,we observed a color change accompanied with reduced light scatteringintensity, which was confirmed by a vanishing count rate at the DLS,indicating dissolution of HPL EbNPs.

1.2 Synthesis and Characterization of IAT EbNPs

FIG. 6 illustrates two IAT EbNP synthesis protocols. IAT EbNPssynthesized in ethylene glycol exhibit pH stability due to favourablestacking of the IAT molecules, while IAT EbNPs synthesized via thewater-based pH drop method dissolve upon pH increase. FIG. 7 shows thediameters of IAT EbNPs synthesized via the water-based pH-drop method asa function of acid added to the system. Here, we added rapidly undervigorous stirring defined amounts of HNO₃ to 10 ml of 0.05 wt % IATsolution at pH 12 inducing a sudden pH drop, which triggered IATprecipitation as EbNPs. The final pH values of the samples are reportedon the top x-axis. Size control could be achieved by appropriatelyadjusting the amount of acid added.

Native IAT EbNPs were synthesized via the organic solvent water-basedpH-drop method in ethylene glycol and the data are reported in FIG. 8.This method allows the synthesis of IAT EbNPs with good size control inthe range of 50 to 125 nm. First, 0.25 g commercial IAT lignin wasdissolved in 50 ml of ethylene glycol, vortexed for 30 minutes, andfiltered with a 0.45 μm syringe filter. Then, 5 ml of the stock solutionwas put into a 20 ml scintillation vial and vigorously stirred with afitting magnetic stir bar. Supersaturation was induced upon addition ofvarious amounts of acid precipitating out negatively charged IAT EbNPs.We diluted the EbNP suspension in ethylene glycol with Millipore H₂O,after 5 minutes and 7 days, to obtain a 0.05 wt % IAT EbNP sample,containing 10% (v/v) of ethylene glycol. The pH values measured forthese final suspensions range from 3 to 4. The EbNPs exhibited anegative ξ-potential with a magnitude of approximately −30 mV for allsamples. The polydispersity widths increase slightly with increasingparticle diameters. The 7 day synthesis shows that the particles aregrowing when kept in ethylene glycol solution. The IAT EbNPs dispersedin water are stable for periods longer than 84 days.

1.2.1 IAT EbNP pH Stability

As reported in FIG. 9, the IAT EbNPs obtained are pH-stable in the pHrange from 3 to 9. Here, we took dialyzed native IAT EbNP suspensions atpH 4.5 and adjusted the pH with NaOH or HNO₃ solution. The z-averagediameter and the ξ-potential of each sample were measured. The colloidalinstability of the IAT EbNP suspensions below pH 3 can be explained withthe drop in ξ-potential indicating decreased electrostatic repulsionbetween the particles. At higher pH values, the decrease in particlesize may indicate dissolution of particles. To quantify the dissolutionof IAT EbNPs at elevated pH, we performed a UV-Vis study to determinethe remaining amount of IAT lignin precipitated as particles as afunction of increasing pH. FIG. 10 shows the UV-Vis calibration curveobtained at a wavelength of 285 nm. We utilized the curve tocharacterize the pH stability of IAT EbNPs as reported in FIG. 11. Westarted with dialyzed IAT EbNPs at pH 4.92 and adjusted the pH valueafterwards. We then took the supernatant of these pre-treated samplesand added aliquots of NaOH solution to baseline the samples. Then, wedetermined the IAT lignin dissolved in the supernatant and closed theIAT mass balance to obtain the mass of IAT EbNPs remaining in thesuspension at each pH value. For example, when we increase the pH of thenative EbNP suspension from pH 4.92 to pH 8.2, 53.8% of the initial IATEbNPs remain in form of EbNPs, while 46.2% of the initial IAT EbNPs aredissolved.

1.2.2 IAT EbNP Ionic Strength Study and DLVO Modeling

We performed an ionic strength study to investigate the effect ofincreasing ionic strength on the stability of IAT EbNP suspensions, andto determine if the IAT EbNP suspensions may exhibit colloidal stabilityat ionic strength levels equivalent to the ones found in physiologicaltesting media used in biocidal testing. FIG. 12 shows the particlediameter and ξ-potential as a function of ionic strength in mol/L. Weadded measured amounts of NaCl to adjust to the target ionic strength−0.10 M NaCl is equivalent to 0.10 mol/L ionic strength. The colloidalstability of the EbNP suspension was confirmed by DLS size measurements.The magnitude of the measured ξ-potential decreases rapidly upon a smallincrease of ionic strength. We observed that the sample at 0.30 mol/L,at an ionic strength well above the one found in physiological testingmedia (0.015 mol/L), exhibited colloidal stability even after 36 days.The EbNP suspensions above 0.30 M NaCl started to show signs ofcolloidal instability in the form of settling and aggregation.

We modeled the interaction energy W(D) according to DLVO theory inselected IAT EbNPs at three chosen ionic strengths. At an ionic strengthof 0.25 mol/L, we determined a Debye length k⁻¹/=0.61 nm. Withξ-potential of −15.0 mV at that ionic strength, we calculated a surfacepotential Ψ₀=−40.8 mV. We determined the electrostatic repulsion energyW(D_(elec)) and the van der Walls attraction energy W(D_(VDW)) as afunction of the separation distance D to evaluate the total interactionenergy W(D). As shown in FIG. 13, the stability threshold in thecolloidal system was determined to be at 0.25 mol/L ionic strength. Thecolloidal suspension at 0.50 mol/L ionic strength shows a highlyunstable sample.

1.3 Characterization Ag⁺ Ion Infused EbNPs

We obtained IAT EbNPs with a hydrodynamic diameter of 72 nm with apolydispersity index of 0.230 and ξ-potential of −23.5 mV. The DLSequipment measured a conductivity of 0.139 mS/cm, and an electrophoreticmobility of −1.403 μm cm/V s. As depicted in FIG. 14, TEM micrographsshow predominantly nanosized non-spherical clusters in the size rangebelow 100 nm. Some degree of spreading of IAT particles on the TEM gridcould be triggered by the hydrophilic nature of IAT. The structures ofthe clusters suggest a high availability of surface area for particlefunctionalization.

We functionalized negatively charged IAT EbNPs with Ag⁺ ions in aqueoussolution. We chose a common soluble salt, AgNO₃, as an Ag⁺ ion source.FIG. 14 illustrates the possible adsorption of Ag⁺ ions on thedeprotonated ionized groups on the EbNP surface. The main functionalgroups of IAT lignin include phenolic —OH, aliphatic —OH, carboxylgroups —OOH, and thiol groups —SH, which when deprotonated render thesurface charge of TAT EbNPs negative; hence, deprotonated functionalgroups serve as suitable binding sites for cations including Ag⁺ ions.The distribution of the functional groups and the respective pKa valuesare reported in Table 6. TEM micrographs of functionalized IAT EbNPsshow predominantly nanosized non-spherical clusters in the size rangebelow 100 nm.

Ag⁺ reference solutions in the range of 0.25 ppm to 500 ppm wereprepared from 1000 ppm Ag⁺ standard, and the corresponding potential inmV was recorded using an Ag⁺ ion selective electrode in conjunction witha multimeter. Each reading was obtained after 2.5 minutes of equilibriumtime. FIG. 15 shows the Ag⁺ ion calibration curve with logarithmic fit.

We prepared additional AgNO₃ standards with 40 ppm Ag⁺, 100 ppm Ag⁺, 200ppm Ag⁺, and 800 ppm Ag⁺, and added 0.5 ml of each of these standards to9.5 ml of previously prepared 0.0526 wt % IAT EbNP suspensions to infusethe particles with Ag⁺ ions. To determine the Ag⁺ ion content adsorbedon the EbNPs, we first determined the residual Ag⁺ ions in thesupernatant in each sample, and then closed the Ag⁺ ion balance toestimate the amount of Ag⁺ ions adsorbed on the particles. The ioncontent in the supernatant was determined with an Ag⁺ ion selectiveelectrode (ISE).

Table 1 summarizes the Ag⁺ ion infused samples with various amounts ofAg⁺ ion loadings. The initial loading corresponds to the overall Ag⁺ ioncontent in the 10 ml sample at the time of infusion. The Ag⁺ ion contentin the supernatant was calculated from the mV reading at the ISE withthe following equation.

${{Ag}_{snat}^{+}\lbrack{ppm}\rbrack} = e^{(\frac{{{ISE}{\lbrack{mV}\rbrack}} + 100.74}{26.105})}$We measured a negative-potential for the Ag⁺ infused IAT EbNPs.

TABLE 1 Ag⁺ adsorbtion data on particles. Adsorbed on Ag⁺ ion EbNP wt %,initial Ag⁺ ion ISE Supernatant particles adsorbed on Ag⁺ ppm loading[mv] [ppm Ag⁺] [ppm Ag⁺] particles [%] IAT 0.05, 2 ppm −98.80 0.91 1.0954.7 IAT 0.05, 5 ppm −64.80 3.58 1.42 28.4 IAT 0.05, 10 ppm −44.30 7.702.30 23.0 IAT 0.05, 40 ppm −5.70 35.07 4.93 12.3

We modeled the Ag⁺ ion adsorption equilibria with a Langmuir adsorptionisotherm and normalized the Ag⁺ uptake capabilities per surface area.The Langmuir adsorption isotherm is described by the following equation:

${\Gamma(c)} = {\Gamma_{\max}\frac{Kc}{1 + {Kc}}}$We determined a maximal adsorption Γ_(max)=8688 ppm Ag⁺/m² particlesurface area, and a K value of 0.000115 in FIG. 16. We used theseparameters to model the adsorption isotherm that we report in FIG. 17.We observed that the Ag⁺ ion loading increases with an increasing amountof Ag⁺ available. The Ag⁺ absorption reaches equilibrium within 24 h. Weobserved that the Ag⁺ content in the supernatant stays constant after 24h (re-measured after 3 days). We infer that the Ag⁺ is predominatelyphysically adsorbed on the IAT binding sites.

1.4 Synthesis and Characterization of Ag-EbNPs-PDADMAC

To allow electrostatic attraction between negatively charged bacteria inaqueous solution and the Ag⁺ ion functionalized EbNPs, we reversed thesurface charge of Ag⁺ infused EbNPs from negative to positive. Wemodified the surface properties of the EbNP system through adsorption ofPDADMAC, a positively charged polyelectrolyte. To find a suitablePDADMAC concentration for the EbNP surface modification, we preparedsamples with the initial PDADMAC concentrations reported in Table 2. Theparticles were coated in 5 ml batches of IAT EbNP 0.05 wt % suspension.For the surface modification step, 5 ml of polyelectrolyte solution withthe previously reported wt % was added rapidly to the IAT EbNPsuspension. The final concentration of IAT in the sample was 0.025 wt %.To investigate the stability and the change of properties of the coatedsamples, we measured the z-averages and the ξ-potential with DLS. FIG.18 illustrated the EbNP diameter and potential trends as a function ofinitial PDADMAC concentration. The results indicate that below additionof 0.10 wt % PDADMAC solution, the IAT EbNP surface potential does notreverse from negative to positive. At 0.15 wt % or higher, enoughPDADMAC is available to reverse the surface charge to positive.

TABLE 2 Initial PDADMAC wt % and resulting ζ-potential afterpolyelectrolyte coating. PDADMAC dispersant) ζ-potential wt % initialwith 5% EG [nm] [mV] Comments 0.050 78.07  38.5 Stable 0.025 73.4  36.4Stable 0.020 73.81  32.9 Stable 0.015 74.65  30.6 Stable 0.010 77.86 23.5 0.005 69.54 −23.0 0.001 75.2 −24.3The magnitude of the positive surface potential, obtained after coatingthe IAT EbNPs, is dependent on the polyelectrolyte used. Similarly tothe samples with PDADMAC coating reported previously, polyallylaminehydrochloride (PAH) coated IAT EbNPs were synthesized to prove thepossibility to customize the surface charge magnitude by choice ofsuitable polyelectrolytes. The z-averages and ξ-potentials were measuredand are reported in Table 3. The corresponding trends are illustrated inFIG. 18.

TABLE 3 Initial PAH wt % and resulting ζ- potential afterpolyelectrolyte coating. z-average (adjusted PAH dispersant) zetapotential wt % initial with 5% EG [nm] [mV] Comments 0.050 79.21 42.8stable 0.020 76.97 42.1 stable 0.010 86.94 38.6 stable 0.005 18.59 13.8unstable

A suitable sample obtained was Ag-EbNPs-PDADMAC (d=72 nm) with a finalIAT EbNP concentration of 0.025 wt %, an Ag⁺ ion content on theparticles of 0.71 ppm, an Ag⁺ ion amount in the supernatant of 1.79 ppm,and a PDADMAC concentration of 0.01 wt % in the colloidal suspension.The surface potential was reversed from −25.0 mV to +32.4 mV with theaddition of PDADMAC −0.01 wt % in the final sample. The final sample pHwas 5.5. Other samples were prepared accordingly.

1.5 Antimicrobial Testing

We compared the antimicrobial activity of Ag-EbNPs-PDADMAC with that ofpositively charged branched polyethylene imine AgNPs (BPEI AgNPs) andAgNO₃ solutions (see supplemental information for BPEI AgNP and AgNO₃sample preparations). We performed quantitative antimicrobial tests onGram-negative E. coli BL21 (DE3), a common human pathogen, andqualitative tests on Gram-negative P. aeruginosa, a human pathogen notsusceptible to antimicrobial amines such as BPEI and PDADMAC. Therefore,any antimicrobial activity in the P. aeruginosa tests will predominantlystem from silver.

The activity of each active agent was determined by comparing the numberof colony forming units (CFU) of a reference plate with the CFU of atest plate as depicted in FIG. 20. The reduction of CFU on a test platewith antimicrobial agent is time dependent and concentration dependent.

The maximum antimicrobial reduction efficiency of 100% was reached whenno CFU could be determined on the test plate. We quantified by theantimicrobial reduction efficiency “E” with the following equation

$E = {100\left( {1 - \frac{{CFU}\mspace{14mu}{sample}}{{CFU}\mspace{14mu}{reference}}} \right)}$

The schematic in FIG. 21 describes the wet method procedure that wasfollowed for antimicrobial tests on both E. coli and P. aeruginosa.First, 200 μl of each active agent was placed into separate lowretention centrifuge tubes. 100 μl of PBS buffer was added to each tubeto baseline the ionic strength, and to adjust the pH value to 7.Finally, 100 μl of bacteria, E. coli or P. aeruginosa solution withapproximately 4400 CFU/ml in nutrient broth, was added. The samples werecontinuously vortexed. After the bacteria were exposed to the activeagent for 1 minute, the survival rate of the bacteria was determined byplating 100 μl of each sample evenly distributed on Luria-Bertani agarplates. The procedure was repeated after 30 minutes of exposure time.After the plating procedure, the petri dishes were sealed and incubatedupside-down for 48 h at 37° C.

1.5.1 Quantitative Antimicrobial Test on E. Coli

FIG. 22 and

Table 4 compare the quantitative antimicrobial efficiency of each activeagent in the E. coli tests. The reduction efficiency of six differentsamples with increasing Ag ppm equivalent ranging from 0 ppm to 54 ppmwas investigated. The graphs show the reduction efficiency at two timepoints, 1 minute and 30 minutes. The weight percentages of the controlsamples and the silver contents in the active agents were chosen to showantimicrobial thresholds and to facilitate comparisons between thesamples. Native IAT EbNPs without Ag⁺ functionalization and surfacemodification did not result in any observable reduction in CFU (notreported), which suggests that the native IAT EbNPs are benign. Also,IAT EbNPs with Ag⁺ functionalization but without PDADMAC coating did notresult in significant reduction of CFU after 1 minute (0%) and 30minutes (5%). We suggest that the low antimicrobial efficiency may beattributed to the negative surface charge of these EbNPs, which mayhinder them from overcoming the electrostatic barrier between theparticles and the bacteria. IAT EbNPs coated with PDADMAC resulted instrong reduction of CFU after an exposure time of 30 minutes, which maybe attributed to the antimicrobial effect of the quarterly aminePDADMAC. PDADMAC solution alone (not reported) exhibited strongbactericidal effects towards E. coli as well, comparable to theefficiency of Ag-EbNPs-PDADMAC or 100% after 30 minutes exposure time.Ag-EbNPs-PDADMAC exhibited strong reduction in CFU, prevalent after 1minute exposure time. The corresponding supernatant of Ag-EbNPs-PDADMACexhibited no observable effect after 1 minute. The reduction of CFU inthe supernatant after 30 minutes exposure time may be explained byresidue active agent in the solution. BPEI AgNPs and AgNO₃ solutionsexhibited antimicrobial effects at 20 ppm Ag and 40 ppm Ag respectively.Overall, the Ag-EbNPs-PDADMAC sample outperformed the BPEI AgNPs andAgNO₃ samples in terms of antimicrobial efficiency normalized on Ag ppmequivalent.

TABLE 4 Results of quantitative E. coli tests. The CFU reductionefficiency of selected IAT EbNP samples, BPEI AgNP samples, and AgNO₃samples are shown. Quantitative E. coli test; IAT EbNP samples snatIAT0.025 IAT0.025 Ag⁺ 2.5 ppm IAT0.025 Ag⁺ 2.5 ppm IAT0.05 PDADMACPDADMAC PDADMAC Ag⁺ 5 ppm 0.01 0.01 0.01 1 min vortex time CFU no 95.24%12.38% no reduction observable observable efficiency effect effect 30min vortex time CFU 4.76% 100.00% 100.00% 89.52% reduction efficiencyQuantitative E. coli test; AgNPs BPEI BPEI BPEI BPEI BPEI AgNPs AgNPsAgNPs AgNPs AgNPs 54 ppm 20 ppm 10 ppm 5 ppm 2.5 ppm 1 min vortex timeCFU 84.76% no no no no reduction observable observable observableobservable efficiency effect effect effect effect 30 min vortex time CFU100.00% 91.43% no no no reduction observable observable observableefficiency effect effect effect Quantitative E. coli test; AgNO3 AgNO3AgNO3 AgNO3 AgNO3 AgNO3 40 ppm 20 ppm 10 ppm 5 ppm 2.5 ppm 1 min vortextime CFU 53.33% no no no no reduction observable observable observableobservable efficiency effect effect effect effect 30 min vortex time CFU96.19% no no no no reduction observable observable observable observableefficiency effect effect effect effect

1.5.2 Qualitative Antimicrobial Test on P. Aeruginosa

As mentioned previously, the qualitative antimicrobial test on P.aeruginosa can distinguish the antimicrobial effect of PDADMAC from theeffect of silver. FIG. 23 and Table 5 compare the qualitativeantimicrobial efficiency of each active agent in this test. BPEI AgNPsand AgNO₃ solutions exhibited no complete antimicrobial effect after 30minutes incubation time at 54 ppm Ag and 20 ppm Ag respectively. Thecontrol sample IAT EbNP at an elevated wt % of 0.10 did not result inany observable effect. Also, the sample IAT EbNPs coated with PDADMACdid not exhibit any measurable antimicrobial effect. The PDADMACsolutions at 0.02 wt % and 0.04 wt % appeared to promote P. aeruginosagrowth. The sample Ag-EbNPs-PDADMAC was the only one that exhibitedcomplete or 100% antimicrobial efficiency after 30 minutes. Thesupernatant of the sample Ag-EbNPs-PDADMAC did not show anyantimicrobial effect after 30 minutes of incubation time. As the controlsamples of PDADMAC 0.02 wt %, PDADMAC 0.04 wt %, and the IAT EbNP samplecoated with PDADMAC were ineffective in terms of complete antimicrobialefficiency after 30 minutes of incubation time, the results suggest thatthe antimicrobial action of Ag-EbNPs-PDADMAC is delivered by silverions. Comparing all active agents tested in terms of antimicrobialefficiency, we establish that Ag-EbNPs-PDADMAC proved most effective.

TABLE 5 Result table of qualitative Pseudomonas aeruginosa test. Eachpicture corresponds to the four reported CFU tests in the table above.After 30 min of vortexing, growth was observed in bacteria treated withBPEI AgNPs 54 ppm, AgNO₃ 20 ppm, PDADMAC 0.04 wt % solution, IAT EbNPs0.10 wt %, IAT EbNPs 0.05 wt % coated with PDADMAC 0.02 wt %, and thesupernatant of 0.05 wt % Ag-EbNPs-PDADMAC. The active agent 0.05 wt %Ag-EbNPs-PDADMAC was the only active agent resulting in no growth or100% reduction in CFU. Qualitative P. aeruginosa test IAT0.05 Ag⁺ snatIAT0.05 IAT0.05 5 ppm Ag⁺ 5 ppm BPEI AgNPs BPEI AgNPs AgNO₃ AgNO₃PDADMAC PDADMAC PDADMAC PDADMAC PDADMAC 10 ppm 54 ppm 10 ppm 20 ppm 0.020.04 IAT0.10 0.02 0.02 0.02 1 min vortex time or contact timePseudomonas Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes aeruginosa growth 30min vortex time or contact time Pseudomonas Yes Yes Yes Yes Yes Yes YesYes No Yes aeruginosa growth

2 CONCLUSION

We developed a new class of nanomaterials with increased efficiency andpotentially improved nanoparticle post-utilization safety.Functionalized environmentally benign nanoparticles (EbNPs) exhibitlocally confined and temporarily limited bioactivity. Other than theirpersistent counterparts, they are predominately made from biodegradableand sustainable materials, and are synthesized via green chemistry. Asthese EbNPs may lose their activity due to depletion of agent,dissolution of the EbNP system, or degradation of the lignin-basedmatrix by the environment, they can minimize any potential nanomaterialwaste hazards. In addition to the beneficial post-utilizationperformance, EbNPs may deliver higher efficiency in terms of activeagent employed in comparison to persistent nanoparticle system. Inbiocidal tests on the human pathogens E. coli and P. aeruginosa, weproved that silver ion infused EbNPs with positive surface charge(Ag-EbNP-PDADMAC) exhibit significantly higher antimicrobial activitiesin terms of Ag equivalent than silver nanoparticles. The increasedefficiency of EbNPs with functional equivalent to their persistentcounterparts, may favor substitution of a wide range of applied metalnanoparticles. Moreover, the benign nature of f-EbNPs opensopportunities for new applications of nanoparticles in the agriculture,home and personal care, and pharmaceutical industry.

3 EXPERIMENTAL SECTION Equipment

DLS (Malvern Instruments Ltd., Nano ZS, λ=633 nm, max. 5 mW)

Syringe pump (New Area Pump Systems, NE-4000)

UV-Vis spectrometer (Jasco UV/Vis V-550 spectrophotometer)

UV lamp (Uvitron, Sunray 400SM)

Multimeter (Mettler Toledo, S80)

Materials and Chemicals Used in EbNP Synthesis.

Lignin.

We obtained INDULIN AT lignin (IAT) powder (lot MB05) and supportingdocumentation from MeadWestVaco (MWV) Charleston, S.C. We estimated thedistribution of the main functional groups per 100 aromatic unitsaccording to the literature provided by MWV, and assigned pKa valuesfrom tables. We obtained High Purity Lignin (HPL) powder and supportingdocumentation from Lignol Burnaby, BC, Canada, and assigned pKa valuesto its functional groups accordingly. Table 6. FIG. 6 shows thedistribution of functionality of the main functional groups of bothlignins.

TABLE 6 Main functional groups of IAT and HPL lignin, and their pKavalues. IAT, distribution of HPL, distribution of Lignin functionalfunctionality functionality groups (#/100 aromatic units) (#/100aromatic units) pKa Phenolic OH 68 73 10 Aliphatic OH 51 34 18 SH 9 —10.75 COOH 16.02 — 4.2 OCH3 82 114  10.2 Carbonyl - O 12 — — Aryl alkylether 36 — — Dialkyl ether 9 — —

Millipore water (Synergy UV); acetone (BDH, CAS#67-64-1, lot 010612B);HNO₃ (Sigma Aldrich, CAS#7697-37-2, lot A0294591); ethylene glycol(Sigma Aldrich, CAS#107-21-1, grade 99+%, lot B0521395); 0.45 μm syringefilter (Thermo Scientific, nylon syringe filter 0.45 μm); magnetic stirbar (Fisher Scientific, 8-Agon stir bar 14-512-147).

HPL EbNP Synthesis.

The ξ-potential was measured with a Malvern disposable capillary cellDTS 1061. The following measuring settings were used: the solvent wasH₂O with 10% (v/v) acetone with an overall viscosity of 1.0684 cP. Theeffective voltage was 150 V.

IAT EbNP Synthesis.

The ξ-potential was measured with a Malvern disposable capillary cellDTS1061 in the size control and pH-stability studies. The followingmeasuring settings were used: the solvent was H₂O with 10% (v/v)ethylene glycol with an overall viscosity of 1.1932 cP. The effectivevoltage was 150 V. The ξ-potential was measured with a Malvern dip cellZEN1002 in the ionic strength study. The dip cell allows ξ-potentialanalysis with low driving voltages. The following measuring settingswere used: the solvent was H₂O. The voltage was automatically adjustedby the equipment and chosen at values below 5.0 V for all measurements.

IAT EbNP Functionalization with Ag⁺ Ions.

Ag⁺ standard (Mettler Toledo, silver ISE standard 1000 ppm 51344770, lotISEAG510L1); Ag⁺ ion selective electrode (Mettler Toledo, silver/sulfurelectrode 51302822, reference filling solution C 51344752).

Reference Samples BPEI AgNPs and AgNO₃ Solution.

Positively charged branched polyethylene imine (BPEI, Sigma Aldrich, Mw˜25000 by LS, CAS#9002-98-6, lot MKB64206V) coated AgNPs with az-average diameter of 20 nm were synthesized according to theliterature.²⁹ The molar ratio of the final AgNP solution was chosen tobe 0.5 mM BPEI: 0.5 mM AgNO₃ (Fisher Chemicals, CAS#7761-88-8, lot016932): 0.1 mM HEPES buffer (Sigma Aldrich, CAS#7365-45-9, lot98H5425). 10 ml of the mixture was exposed to UV light for 120 minutesto form BPEI capped AgNPs with 54 ppm silver equivalent. The z-averagediameter was determined with DLS. The pH value of the final solution was6.3. AgNO₃ solutions were prepared from a 1000 ppm Ag⁺ referencestandard. The target ppm concentrations for antimicrobial testing werereached by appropriately diluting the reference standard with Milliporewater.

Media Used in Antimicrobial Testing.

PBS buffer (Sigma Aldrich, CAS#7778-77-0, lot 38H8503), LB ager (FischerChemicals, CAS#9002-18-0), LB broth (Acros 61187-5000, lot B012260G).

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Section 2

As depicted in FIG. 1, in contrast to permanent nanoparticle systemsexhibiting post utilization hazards for humans and the environment, dueto nanoparticle migration, accumulation, and persistent activity, theobject of the present invention—functionalized EbNPs—have increasednanomaterial safety as they will biodegrade after their intended useminimizing any hazardous effects stemming from nanomaterial waste. Meansof synthesizing native EbNPs from biopolymers via green synthesismethods in simple, inexpensive, and non-toxic ways are illustrated.Functionalization options via adsorption of and/or infusion with activeagents are described. A range of procedures to alter the EbNP pHstability, colloidal stability in water or organic media, and surfaceproperties have been developed. Silver-infused EbNPs with positivesurface charge capable of releasing antimicrobial silver ions weresynthesized, and evidence for complete functional equivalency tohigh-volume nanoparticles currently employed in industry—antimicrobialsilver nanoparticles (AgNPs)—was demonstrated. In biocidal tests onhuman pathogens such as E. coli BL21(DE3) and Pseudomonas aeruginosa,the EbNPs showed higher efficiency in comparison to AgNPs, and silvernitrate (AgNO₃) solution. While the native EbNPs did not exhibit anyobservable biocidal activity, positively charged EbNPs infused withsilver ions demonstrated significantly higher antimicrobial efficiency.In addition to the beneficial performance with less active agentfavoring substitution of AgNPs and others with EbNPs with functionalequivalency, the benign nature of the invention opens opportunities fornew applications in safety sensitive applications including the food anddrug industry.

The invention can be used as a platform technology for versatilesynthesis of functionalized EbNPs, in achieving functionality andefficiency, in formulation, and in applications, and environmentalsafety and biodegradability at the same time. These EbNPs are systemizedfrom natural materials available in abundance, potentially from waste orbio-products streams, by employing simple processes using green or noorganic solvents. In addition to their safety, efficiency, and costadvantages, the benign nature of the new EbNPs opens multipleutilization opportunities in markets closed for persistentnanoparticles. The advantageous features of the invention include:

-   -   Process novelty: The synthesis procedures for persistent        nanoparticle systems often involve hazardous materials, complex        methods, and reactions at elevated or reduced temperatures, but        only yield small output volumes/yields. Functionalized        environmentally benign nanoparticles (EbNPs) are (1) made of        sustainable materials and synthesized via novel, green, simple,        and scalable methods, (2) functionalized with active agent,        and (3) surface modified to achieve complete functional        equivalency. Large yields and unmatched cost advantage in        comparison to a variety of permanent and hazardous metal or        semiconductor nanoparticles can be achieved.    -   Functionality and Efficiency: Nanoparticles made entirely of        active agent may not make efficient use of the agent.        Functionalized EbNPs are primarily made of sustainable        materials. The EbNP surface-active functionalization agent can        be released more easily increasing the efficiency of the agent.        Surface property modification e.g. to protect the active agent,        to alter the release properties of the active agent, to change        the stability of the system, to alter attractive or repulsive        forces to a specific target, to change the charge or        hydrophobicity of the system, and to prepare the system for        further functionalization and/or modification serve as further        possibilities to optimize the efficiency of the EbNPs. As a        result, functionalized EbNPs use in general significantly        smaller amounts of active agent to deliver the same        functionality than their persistent counterparts.    -   Additional Safety: Hazardous risks associated with persistent        nanoparticle waste have been recognized by the Environmental        Protection Agency (EPA)^(30, 31). Other than persistent        nanoparticles exhibiting post utilization hazards for humans and        the environment, due to nanoparticle migration, accumulation,        and activity, functionalized EbNPs increase nanomaterial safety        as they will deplete rendering the particles benign, which        biodegrade after their intended use minimizing any hazardous        effects stemming from nanomaterial waste.    -   Formulation novelty: Choice of materials in functionalized EbNPs        results in specific novel ingredient formulations.    -   Application novelty: Persistent nanoparticle systems have        limited application potential. The benign nature (by design) of        the invention opens usage opportunities in markets closed for        persistent nanoparticles, which can be found in the multibillion        pesticide, food, and drug industries.

Functionalized EbNPs consisting of a core, one or more functionalizingagents, and one or more surface modifiers, whose details are outlined inthe definition of terms, could be based on the following combination ofmaterials. The EbNP core consists mainly of biopolymers or a combinationof biopolymers from the group of modified lignin, modified cellulose,hemi or lingo cellulose, modified chitosan, modified chitin, prolamines,and gluten. Most preferable lignins are modified lignins extracted viaKraft pulping process including sulfonated (INDULIN C lignin) andunsulfonated lignin (INDULIN AT lignin) from MeadWestvaco and others,and extracted via organosolv process such as High Purified Lignin(HP-L™) from Lignol. Preferable modified lignins include Kraft pulpinglignin extracted via Lignoboost process from Metso, and theirderivatives. Most preferable modified celluloses include hydroxyl propylmethyl cellulose phthalate (e.g., HP-55 or HP-55S from Shin-Etsu),hypromellose acetate succinate (e.g., AS-HF from Shin-Etsu), and theirderivatives. Other preferable modified celluloses include methylcellulose, ethyl cellulose, cellulose acetate, hydroxyethyl cellulose,cellulose nitrate, cellulose propionate, and their derivatives. Mostpreferable modified chitosans include low molecular weight (Mw)chitosans, and their derivatives including chemically modified chitosanwith amino groups in the backbone of chitin. Preferable chitosansinclude medium and high Mw chitosan and their derivatives. The activeagent may be any biologically active component. This includes mostpreferably monovalent and divalent cationic metal ions includingbiocidal Ag⁺ and Cu²⁺, biocidal semiconductor compounds such as ZnO andTiO₂, and natural and synthetic pesticides. Preferable active agents areamines, biopolymers with biocide activity including low Mw chitosan withamino groups, and synthetic polymers including negatively and positivelycharged polyelectrolytes. Other possible functionalization agentsinclude the group of hydrophilic and hydrophobic pharmaceuticals, foodadditives, proteins, peptides, and others. Surface property modifiersare surfactants that include synthetic polymers and biopolymers asdefined previously. Most preferably are positively charged biocidalamines such as polydiallyldimethylammonium chloride (PDADMAC) orpoyallylamine hydrochloride (PAH), and oils and silicon basedsurfactants used as pesticide transfer and targeting agents. Othergroups of surface modifiers include positively and negatively chargedpolyelectrolytes for surface charge modification, non-ionic surfactants,protein based surfactants, emulsifiers, and polysaccharides.

Negatively charged hydrophobic EbNPs are synthesized via one of the foursuggested green synthesis routes with mean diameters most preferably inthe range of 20 to 100 nm. Preferable EbNPs from synthesis may alsoresult in bigger particles with mean diameters typically up to 500 nm,or more. The procedures include the water-water based pH-drop method,the solvent-water based pH-drop method, solvent-antisolvent method, andthe polyelectrolyte-addition method. Taking lignin as input material,INDULIN AT lignin (IAT) and HP-L™ nanoparticles have been synthesized bythe previously mentioned procedures. Table 7 compares the advantages andlimitations of each method. In the water-water based pH-drop method,supersaturation and subsequent nanoparticle formation are achieved uponaddition of acid to dissolved lignin in water at elevated pH to drop thepH into the range of pH 1.5 to 3.0. The pH stability can be increasedupon adsorption of positively charged polyelectrolytes most preferablywith PDADMAC, PAH, and others on the negatively charged EbNP surface. Inthe organic solvent-water based pH drop method, lignin is firstdissolved in organic solvent such as ethylene glycol, toluene, orsimilar. Supersaturation is reached upon addition of acid precipitatingout negatively charged hydrophobic EbNPs. The EbNPs formed in organicmedia may be transferred into water via dialysis or dilution. In thesolvent-antisolvent method, biopolymer is dissolved in solvent such asacetone or ethanol. Supersaturation is reached upon rapid addition ofantisolvent such as water precipitating out negatively chargedhydrophobic EbNPs. In the polyelectrolyte-addition method, biopolymer isdissolved in solvent, typically water at adjusted pH. EbNPs are formedupon addition of positively charged polyelectrolytes. In comparison toprior art, each of the four green synthesis routes is performed at roomtemperature without crosslinking reaction. This differs significantlyfrom the methods reported in patent literature in which chemicallymodified cross-linked lignin nanoparticles were synthesized at elevatedtemperatures^(32, 33). While chemically modified lignin nanoparticlesmay not biodegrade as easily, other advantages of the green synthesismethods described include utilization of inexpensive materials, lowhazard potential, room temperature operations and therefore no need forexternal energy input or cooling, size control, scalability, and shortsynthesis times from prepared stock solutions to synthesized EbNPs inthe minute range. According to the advantages outlined, the EbNPsynthesis costs are low.

The second part of the invention includes nanoparticle functionalizationin order to infuse the matrix with an active ingredient or otherwisecreate the desired usage characteristics. Functionalization methods ofthe EbNP carrier include infusion, and absorption and/or physical andchemical adsorption of active agent. Both weak and strong binding of theactive agent are possible mechanisms involved in the functionalization.This binding of the agent can occur because of electrostaticinteraction, hydrophobic or hydrophilic interactions, reductionprocesses, chemical linking, kinetic and entropy driven capture of thefunctional molecules. In comparison to persistent nanoparticle systemscompromised of the active agent alone, the functionalized EbNPtechnology suggests higher efficiency in terms of optimized smalleramount of active agent used to deliver the same functionality ultimatelyminimizing risks and hazards stemming from excess active agent.

The adjustment and customization of the surface properties is used toreplicate and enhance the particle properties needed for itsfunctionality, and can be achieved by introducing one or more modifierson the EbNP surface. Depending on the surface modifier chosen, thebinding strength and the adsorption processes can vary accordingly.Surface properties that are controlled on this stage include surfacecharge, pH stability, hydrophobicity, biocidal activity, and others.Changes in surface properties can specifically be performed for betterparticle targeting, to increase the shelf life of the system, to alterthe colloidal stability, to modify the interaction potential with theenvironment or a specific target, to protect the active agent, tocustomize depletion and transport effects of active agent, and others.

Applications:

The EbNP systems suggested in the present invention can findapplications in different areas of technology and industrial products.The key new element is that the functionalized EbNPs may be designed toexhibit locally confined and temporarily limited bioactivity, bydelivering the same desired activity as permanent nanoparticlescurrently employed in various applications, but only during the time oftheir application. Since functionalized EbNPs can be engineered to havecomplete functional equivalency to a variety of permanent hazardousnanoparticles, EbNPs may therefore replace a wide range of metal orsemiconductor nanoparticles employed at moderate temperatures.

Besides these multimillion dollar applications, the benign nature of theinvention opens opportunities for its use in markets presently closedfor persistent nanoparticles. New additional applications may be foundin the multibillion pesticide, food, and drug industries. Applicationsof functionalized EbNPs are sectioned in immediate applications, newapplications, and new applications with FDA approved EbNP matrix.

Immediate applications of these new particles include/invention can beemployed as:

-   -   a) Functionalized environmentally benign nanoparticles (EbNPs)        with antimicrobial properties to be used as additive to        detergents or soaps to establish or increase the antimicrobial,        antifungal, or antiviral function of the product.    -   b) Surface coatings made of biodegradable components consisting        of modified lignin and/or modified cellulose, hemicellulose or        chitin functionalized with positively charged polyelectrolytes        and/or Ag⁺ ions adsorbed in/on the matrix for antimicrobial,        antifungal, and antiviral surface functionalization in consumer        products.    -   c) Functionalized EbNP suspensions for dipping or spraying with        added modified lignin and/or modified cellulose, hemicellulose        or chitin in diameters ranging from 20 to 500 nm, optionally        functionalized with positively charged polyelectrolytes, and/or        Ag ions adsorbed on the particles, in water-based or organic        solvent-based solution, for antimicrobial, antifungal, and        antiviral functionalization of surfaces.    -   d) Functionalized EbNPs consisting of modified lignin and/or        modified cellulose, hemicellulose or chitin in diameters ranging        from 20 to 500 nm functionalized with positively charged        polyelectrolytes and/or Ag ions adsorbed on the particles to be        incorporated in/adsorbed on general available water filtration        matrixes to provide antimicrobial, antifungal, and antiviral        water treatment in addition to basic water filtration.    -   e) EbNPs consisting of modified lignin and/or modified        cellulose, hemicellulose or chitin in diameters ranging from 20        to 500 nm functionalized with biocidal agents such as Ag⁺, Cu²⁺,        or iron oxides coated with positively charged polyelectrolyte        incorporated in/adsorbed on general available water filtration        matrixes to provide antimicrobial, antifungal, and antiviral        water treatment in addition to basic water filtration.    -   f) Functionalized EbNPs to be used as general active agent        carrier system specifically to substitute silicon based particle        systems used in the pesticide and food industry.    -   g) EbNPs functionalized with pesticides and coated with        pesticide transfer agent to be used as benign pesticide agent        carrier and delivery system with engineered temporal activity to        reduce the amount of active agent used and therefore any        negative environmental impact while delivering the same        functionality, and to increase product safety for humans.    -   h) Functionalized EbNPs in diameters ranging from 20 to 500 nm        functionalized with positively charged polyelectrolytes and/or        Ag⁺ ions adsorbed on the particles to be incorporated in and/or        adsorbed on general types of water filtration matrixes to        provide antimicrobial, antifungal, and antiviral water treatment        in the pharmaceutical industry.    -   i) Nano-carriers made of biopolymers in diameters ranging from        20 to 500 nm functionalized with positively charged        polyelectrolytes and/or Ag⁺ ions adsorbed on the particles to be        incorporated in and/or adsorbed on general types of water        filtration matrixes to provide antimicrobial, antifungal, and        antiviral water treatment in the food industry.    -   j) EbNPs in diameters ranging from 20 to 500 nm functionalized        with biocidal agents such as Ag⁺, Cu²⁺, or iron oxides coated        with positively charged polyelectrolyte incorporated in/adsorbed        on general available water filtration matrixes to provide        antimicrobial, antifungal, and antiviral water treatment in        addition to basic water filtration.    -   k) EbNPs functionalized with active agent to be used as general        active agent carrier system specifically to substitute        silicone-based particle systems used in the pesticide industry.    -   l) EbNPs functionalized with pesticides and coated with        pesticide transfer agent to be used as benign pesticide agent        carrier and delivery system with engineered temporal activity to        reduce the amount of active agent used and therefore any        negative environmental impact while delivering the same        functionality, and to increase product safety for humans.    -   m) EbNPs functionalized with organic pesticides and coated with        pesticide transfer agent to be used as benign pesticide agent        carrier and delivery system with engineered temporal activity to        reduce the amount of active agent used and therefore any        negative environmental impact while delivering the same        functionality, and to increase product safety for humans.    -   n) EbNPs functionalized with antimicrobial agent to be used as        benign antimicrobial agent carrier and delivery system with        engineered temporal activity to combat bacterial and fungal        plant pathogens to reduce vegetable spoilage.    -   o) EbNPs functionalized with antimicrobial agent to be used to        functionalize dressings and tampons with antimicrobial effect to        decrease the potential of infections.    -   p) EbNPs functionalized with antimicrobial agent to be used to        functionalize an article of hygiene with antimicrobial        properties to decrease the potential of infections.    -   q) EbNPs functionalized with antimicrobial agent to be used to        functionalize paper with antimicrobial properties to decrease        the potential of infections.

New applications include:

-   -   r) EbNPs consisting of modified lignin and/or modified        cellulose, hemicellulose or chitin in diameters ranging from 20        to 500 nm, optionally functionalized with positively charged        polyelectrolyte, to be used as adsorption matrix for monovalent        and divalent hazardous metal ions and hydrophobic agents in        liquid-based environmental remediation processes.    -   s) Process of making EbNPs consisting of modified lignin        synthesized via solvent-antisolvent (where water could be both        solvent and anti-solvent) process from organosolv lignin, or        synthesized via pH drop method in organic solvent or water, or        synthesized via addition of negatively charged polyelectrolyte        to lignin dissolved in solvent, and/or modified cellulose        synthesized via pH drop method in organic solvent or water, or        synthesized via addition of negatively charged polyelectrolyte        to cellulose dissolved in solvent, in diameters ranging from 20        to 500 nm, optionally functionalized with positively or        negatively charged polyelectrolyte, to be used as        environmentally benign fillers and protectors in paints in the        micro and nano size range in wood constructions.    -   t) EbNPs functionalized with biocides to be used as paint        additive to decrease susceptibility of growth of prokaryotic or        eukaryotic cells on the surface.    -   u) EbNPs functionalized with biocides to be used as additives in        sound or heat isolation materials or fillers to decrease        susceptibility of growth of prokaryotic or eukaryotic cells on        the surface or in the bulk of such materials.    -   v) Hydrophobic EbNPs with optional biocidal functionalization to        be used in wood protection and/or as paint additive to decrease        both the wettability of the product and the growth of        prokaryotic and eukaryotic cells on the surface of wood products        to increase their lifetime.    -   w) EbNPs to be used as binder additive into paints, ceramics,        glues, or coating materials dispersed in the product to serve as        linker between the components upon drying increasing the        stability of the product.    -   x) Functionalized EbNPs to be gathered in porous particles frits        to serve as catalyst facilitating chemical reactions.    -   y) EbNPs functionalized with catalytic metals such as Pt or Pt        alloys immobilized in porous frits to serve as catalysts        facilitating chemical reactions.    -   z) Functionalized EbNPs with surface coating with specific        adsorption potential to serve as adsorption material for        targeted biomolecules to be used in biosensor applications.    -   aa) Bioassays using EbNPs with possibly additional marker or dye        functionalization with surface coating exhibiting adsorption        potential to specific biopolymers to be used in biomolecular        screening applications.    -   bb) Functionalized EbNPs to be used on bioassay microchip to        serve as biomolecule specific adsorption material.    -   cc) Functionalized EbNPs consisting of synthesized EbNPs and at        least one polyelectrolyte to be used as coating material to        alter surface properties including hydrophobicity, surface        charge, and optional anisotropy.    -   dd) Foam or emulsion based products, where bubbles or droplets        are fully or partially stabilized with EbNPs, having at least        one of the additional functionalities as described above, where        the additional functionality of the particles is boosted (in        terms of dose or efficiency) due to their attachment of the        interface and their benign properties further improved when        these foams or emulsions are destabilized.

New therapeutic or nutraceutical applications (possibly for FDA-approvedor other regulatory approval) of EbNPs described herein include:

-   -   ee) Functionalized EbNPs with biocide functionalization to be        employed as coating material in food containers, bottles, or        cans to decrease antimicrobial or antifungal contamination        potential to protect the food from subsequent spoiling.    -   ff) Functionalized EbNPs with biocide functionalization to be        used as food additive to increase the shelf life through making        the food less susceptible for bacterial or fungal contamination,        and to decrease spreading of bacteria and fungi in the product.    -   gg) Functionalized environmentally benign nanoparticles        consisting of modified lignin and/or modified cellulose,        hemicellulose or chitin in diameters ranging from 20 to 500 nm        to adsorb hydrophobic drugs and beneficial cationic ions and        molecules prior to optional protective functionalization with        positively and negatively charged polyelectrolytes to be used as        transport agent in oral drug delivery.    -   hh) Functionalized EbNPs with antimicrobial properties added as        active agent in disinfecting and cleaning solutions for soft and        rigid contact lenses to protect the eyes of the contact lens        wearer from infections.    -   ii) Functionalized EbNPs with insecticide, herbicide, or        fungicide functionalization to be employed as pesticide in the        agriculture sector to protect the pesticide from dilution and        depletion, to specifically target the pest, and to increase the        pesticide concentration locally at the target while decreasing        the overall average exposure of pesticide on the protected good.    -   jj) EbNPs with marker or dye functionalization to be employed as        a diagnostic or an indicator for exposure routes in humans,        animals, and the environment in general to protect the marker        from dilution and depletion, to decrease the overall indicator        concentration while using a benign matrix.    -   kk) EbNPs functionalized with antibiotics to be delivered in        humans, animals, and the environment in general to protect the        agent from dilution, depletion, and degradation, to decrease the        overall antibiotic exposure concentration while using a benign        matrix.    -   ll) EbNPs with Ag infusion to be used as wound dressing, and        immobilized antibacterial, antifungal, anti-inflammatory active        agent on material used as wound cover.    -   mm) EbNPs with Ag infusion to be incorporated in hydrogels to        provide wound dressing with antibacterial, antifungal, and        anti-inflammatory properties.    -   nn) Functionalized EbNPs with antimicrobial agents to be used as        coating materials of catheters including catheters used in        neurosurgery for cerebrospinal fluid drainage to reduce the risk        of infection.    -   oo) Functionalized EbNPs with antimicrobial agents to be used as        additive to bone cement and implantable materials to reduce        risks of infections.    -   pp) Ag functionalized EbNPs with beneficial properties towards        treatment of dermatitis to be used as in cures resulting in        reduction of dermatitis due to Ag ions stemming from EbNP        system.    -   qq) Ag functionalized EbNPs with beneficial properties towards        treatment of acne resulting to be used in acne treatment        solutions in reduction of acne due to Ag ions stemming from EbNP        system.    -   rr) Functionalized EbNPs with antimicrobial properties to be        used in urology as coating material for surgical mesh to provide        protection towards bacterial infections for pelvic        reconstruction.    -   ss) Functionalized EbNPs (in one non-limiting embodiment, Ag⁺)        with antiviral properties to be used as virus replication        inhibitor in direct virus treatments, and in vaccines.    -   tt) EbNPs functionalized with bactericidal and antifungal agents        to be used as preservatives in vaccines to protect the vaccine        agent from contamination and therefore, the person vaccinated        from infections related to contaminations.    -   uu) EbNPs functionalized with bactericidal and antifungal agents        to be used as coating material in vials, vessels, or syringes        holding vaccines to protect the vaccine agent from contamination        and therefore, the person vaccinated from infections related to        contaminations.

DEFINITION OF TERMS

-   -   Biopolymers include linear, branched, or cross-linked        biopolymers of natural or technical origin (i.e. physically or        chemically modified during processing of natural compounds and        appearing usually as bi-product or waste stream of such process)    -   Biopolymers include large molecular weight natural or technical        polyphenols or natural phenolic polymers like lignin or modified        lignin or a combination thereof.    -   Biopolymers from natural or technical origin include physically        or chemically modified biopolymers during processing of natural        compounds that appear usually as bi-product or waste stream of        such process.    -   Biopolymers include byproducts of degradation of lignin, such as        humic acid and others.    -   Organic solvents include acetone, ethylene glycol, propylene        glycol, glycerol, methyl acetate, ethyl acetate, methanol,        ethanol, hexanol, petrol ether, toluene, benzene, turpentine,        dichlormethane, chloroform, formic acid, isopropanol,        polyethylene oxide, and others.    -   Prolamines include gliadin from wheat, hordein from barley,        secalin from rye, zein from corn, kafirin from sorghum or avenin        from oats.    -   Gluten include gladin and glutenin    -   Cationic metal ions include Ag⁺, Ag²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺,        others, and their complexes; precursor of metal ions for e.g.,        Ag⁺ are salts such as AgNO₃.    -   Anionic ions include anionic antimicrobial peptides and proteins        (AAMPs).    -   Cytotoxic agents include but are not limited to antibacterial        agents such as antibacterial peptides or proteins. Examples        include, but are not limited to,        bactericidal/permeability-increasing protein (BPI); helical        linear peptides, e.g., gramicidin, magainins, melittin; or        β-sheet or cyclic peptides such as defensins, amphotericins and        nystatin.    -   Semiconductors include ZnO, ZnSe, ZnS, ZnTe, CuCl, Cu₂S, CuO,        TiO₂, CdSe, CdS, CdTE FeO, Fe₃O₄, (α,β,γ,ε), Fe₂O₃, Fe₂S, NiO,        Ag₂S, and others.    -   Pesticides include algaecides, avicides, bactericides,        fungicides, insecticides, miticides, molluscicides, nematicides,        rodenticides, glyphosate, and virucides.    -   Amines include primary, secondary, tertiary, and quarternary        structures. This includes polydiallyldimethylammonium chloride        (PDADMAC), poyallylamine hydrochloride (PAH), polyethyleneimine        (PEI), branched polyethyleneimine (BPEI), polyethoxylated tallow        amine (POEA), and others.    -   Non-ionic surfactants include alcohol ethoxylates, alcohol        ethoxyfulfates, and others.    -   Silicon based surfactants include silicone and silicone blends        with polysiloxane chains, and include commercial products such        as Sylgard® 309 (Wilbur-Ellis Company), Freeway® (Loveland        Industries), Dyne-Amic® (Helena Chemical Company), and Silwet        L-77® (Loveland and Helena), and others.    -   Oils include vegetable oil, methylated vegetable oil, seed oils,        crop oils, petroleum based oils, silicon based oils, and blends        of these. Commercial products include MSO® Concentrate        Methylated Seed Oil (Loveland Industries), Hasten® (Wilbur-Ellis        Company), Improved JLB Oil Plus (Brewer International),        Cide-Kick and Cide-Kick II (Brewer International), Syl-tac™        (Wilbur-Ellis Company), Phase™ (Loveland Industries), Agri-dex®        (Helena Chemical Co. or Setre Chemical Co.), Red-Top Mor-Act®        (Wilbur-Ellis Company), and others.    -   Other surface modification and activation agents include        amphiphilic proteins, soy proteins, proteins, DNA, fluorescence        DNA, peptides, fluorescence markers, amino acids, and others.

TABLE 7 Green synthesis methods comparison table. EbNP d pH- Mainparameters for Method material [nm] stability particle size controlAdvantage Limitations water-water IAT, 20- 1.8- increasing pH-dropmagnitude results completely water size control at target pH pH-dropHP-L ™ 200(+) 3.0 in bigger particles based pH stability (can beovercome) higher initial wt % results in bigger native EbNPs notsuitable for particles infusion with active agent organic IAT 20- 3.0-increasing pH-drop magnitude results pH stability organic solventresidues solvent-water 500 10.0 in bigger particles size control (can beminimized relatively pH-drop higher initial wt % results in biggerphysical adsorption of easy) particles cations possible solvent- HP-L ™20- 3.0- increasing dilution rate with antisolvent pH stability organicsolvent residues antisolvent 200(+) 10.0 results in smaller particlessize control (can be minimized) higher initial wt % results in biggerphysical adsorption of cations particles limited polyelectrolyte IAT,20- 3.0- ratio lignin to polyelectrolyte completely water native EbNPsnot suitable for addition HP-L ™ 200(+) 10.0 influences particle sizeand surface based infusion with active agent charge

Section 3

Executive Summary.

One of the most widely used classes of nanomaterials today is the silvernanoparticles (AgNPs), which exhibit general antimicrobial, antisporaland antifungal activity, while being of low toxicity to humans. Theapplication of Ag nanoparticles, however, has met a number of seriousproblems, due to their relatively large cost and the rapidly growingconcerns about the environmental and human dangers by the persistentnanoparticles released post application. We have pioneered a set of newideas that resulted in the demonstration of a novel class offunctionalized, environmentally-benign, nanoparticles (EbNPs) as highlyefficient microbicidal substitutes of the AgNPs (FIG. 24). Theseparticles are made of biodegradable and environmentally benignbiopolymers such as lignin, and are infused with an optimal amount ofsilver in the form of adsorbed Ag⁺ ions. The active Ag⁺ ions arereleased only during the targeted adsorption of thepolyelectrolyte-coated particles onto bacterial targets. We have shownthat the bactericidal action of these silver-loaded, surfacefunctionalized particles exceeds the one of the common Ag nanoparticlesand that the new EbNPs are capable of killing a broad spectrum ofmicrobes. A more detailed description of these preliminary results isprovided in the main section of this document. The nanoparticles willbecome depleted of Ag⁺ ions shortly after use rendering the nanosystembenign. If the EbNPs are released in the environment concerns related tonanowaste toxicity are minimized as the particles will degrade, for theyare made from natural biopolymers. Both the nanoparticles and theprocesses for their fabrication are simple and inexpensive. Thisapproach is not limited only to bactericidal silver ions as activeagent, but can be applied to the development of many new biopolymerparticles with antiviral, antifungal, antitoxin, anticancer, chemicaldecontamination and other functionalities.

Background.

Engineered nanoparticles are in use or being considered for use across awide range of fields. However, lingering concerns about the potentialhealth hazards of nanoparticles and their accumulation in theenvironment have limited the expansion of nanoparticle technology intomany medical, environmental, and military applications. Silvernanoparticles (AgNPs) have emerged as the most widely employedpersistent nanoparticles (PNPs), as their broad-spectrum antimicrobialproperties allow them to combat bacteria strains exhibiting antibioticresistance,¹¹ which are reported in human pathogens includingEscherichia coli (E. coli)¹² , Pseudomonas aeruginosa (P. aeruginosa)¹³,and others. Furthermore, AgNPs are effective agents against viruses(e.g. herpes), spores (e.g., anthrax), and fungi (e.g. Candidaalbicans)³⁴. Such broad-spectrum biocidal agents can minimize thepotential for nosocomial infections¹⁵ in applications includingantimicrobial wound dressings, textiles,²⁰ and water filters.²¹ However,toxicity studies on AgNPs in contact with skin, specifically incommercially available wound dressings, indicate that AgNP containingsolutions exhibit stronger cytotoxic effects toward keratinocytes thando PNP free counterparts.²³ In addition, in vitro studies on mammalianfibroblasts have revealed that AgNP can induce apoptosis.^(24, 25) Inthis context, AgNP may potentially affect human health.²⁶

The environmentally benign nanoparticles (EbNPs) that we have developedaddress these safety concerns associated with nanosystems withoutsacrificing the powerful nano-scale functionality. Biopolymers such aslignin serve as suitable matrix for benign nanoparticle systems. Ligninis the most abundant aromatic polymer in nature,⁴ has an amorphousstructure, and is biodegradable. Matrixes of INDULIN AT lignin (TAT),depicted in FIG. 25, have shown high adsorption capabilities of heavymetal ions for environmental remediation purposes. In theseapplications, cationic metal ions are electrostatically attracted toIAT, which is negatively charged due to deprotonation of its mainfunctional groups.^(8,9) Recently, we reported the synthesis ofpH-stable IAT-based environmentally biodegradable nanoparticles inethylene glycol by a process that is simple, predominantly water-based,and does not include harsh organic solvents or chemical agents.³⁵

We further proved that by infusing IAT EbNPs with functional metal ions,such as antimicrobial silver ions, and additional surface modification,such as switching the surface charge from negative to positive, it ispossible to synthesize degradable nanoparticles that match thenanoparticle functionality of their respective PNPs, while increasingutilization and post-utilization safety. The schematic in FIG. 25 showsthe three steps involved in generating sustainable antimicrobial EbNPs.First, we synthesize negatively charged IAT EbNPs suitable forfunctionalization with cationic metal ions. In the next step, we adsorbantimicrobial silver ions from water solution of silver nitrate.Finally, we reverse the surface potential of the particles from negativeto positive via adsorption of a positively charged polyelectrolyte. Weuse a relatively benign multifunctional cationic polyelectrolyte,polydiallyldimethylammonium chloride [PDADMAC] as illustrated in FIG.25, which has been frequently employed in environmental applicationssuch as water treatment, in consumer products such as cosmetics, and inbiological application including insecticides and algaecides.³⁶ Besidesthe surface charge modification, the PDADMAC layer may protect theparticle system from unintended Ag⁺ ion depletion. In addition, asquarternized amines are known to exhibit antimicrobial effects, thusPDADMAC may potentially increase the antimicrobial efficiency of theEbNPs.³⁷ Hence, the antimicrobial EbNPs (Ag-EbNPs-PDADMAC) withfunctional equivalency to AgNPs consist of (1) a biodegradable EbNPcore, (2) highly antimicrobial silver ions as active agent, and (3) asurface charge modifier. At contact with the cells, the particles canrelease antimicrobial silver ions, which can be transfer into the cell,to perform the desired antimicrobial function leading to bacteria celldeath. In contrast to the metallic silver in AgNPs, silver inAg-EbNPs-PDADMAC is already available in its ionic form and therefore,may be transferred to the cell more readily. This process may result inrapid silver ion depletion of the Ag-EbNPs-PDADMAC system. At the end ofthe lifecycle, the Ag-EbNPs-PDADMAC system, which is depleted of silverions, is rendered inactive and will degrade over time.

Antimicrobial Testing and Comparison of AgNPs with Silver-Infused EbNPs.

We compared the antimicrobial activity of Ag-EbNPs-PDADMAC with the oneof positively charged branched polyethylene imine AgNPs (BPEI AgNPs) andAgNO₃ solutions, We performed quantitative antimicrobial tests onGram-negative E. coli BL21 (DE3), a common human pathogen, andqualitative tests on Gram-negative P. aeruginosa, a human pathogen notsusceptible to antimicrobial amines such as BPEI and PDADMAC. Therefore,any antimicrobial activity in the P. aeruginosa tests will predominantlystem from silver. The testing procedure are reported in Section 1.

The quantitative antimicrobial efficiencies of each active agent in theE. coli tests are compared in FIG. 26. The microbicidal efficiency ofsix different samples with increasing Ag ppm equivalent ranging from 0ppm to 54 ppm was investigated. The graphs show the reduction efficiencyat two time points, 1 minute and 30 minutes. The weight percentages ofthe control samples and the silver contents in the active agents werechosen to show antimicrobial thresholds and to facilitate comparisonsbetween the samples. Native IAT EbNPs without Ag⁺ functionalization andsurface modification did not result in any observable reductions in CFU(not reported), which suggests that the native IAT EbNPs are benign.Also, IAT EbNPs loaded with Ag⁺ but without PDADMAC coating did notresult in significant reduction of CFU after 1 minute (0%) and 30minutes (5%). We believe that the low antimicrobial efficiency may becontributed to the negative surface charge of these EbNPs, which mayhinder them from overcoming the electrostatic barrier between theparticles and the bacteria. IAT EbNPs coated with PDADMAC resulted instrong reduction of CFU after an exposure time of 30 minutes, which maybe attributed to the antimicrobial effect of the quarterly aminePDADMAC. The Ag+-loaded and surface-functionalized sample,Ag-EbNPs-PDADMAC, exhibited strong reduction in CFU, prevalent after 1minute exposure time. The corresponding supernatant of Ag-EbNPs-PDADMACexhibited no observable effect after 1 minute. BPEI AgNPs and AgNO₃solutions exhibited antimicrobial effects at 20 ppm Ag and 40 ppm Agrespectively. Overall, the Ag-EbNPs-PDADMAC sample outperformed the BPEIAgNPs and AgNO₃ samples in terms of antimicrobial efficiency normalizedon Ag ppm equivalent.

TABLE 8 Comparison of conventional AgNPs with the new silver-infusedEbNPs. Parameter Silver Nanoparticles (AgNPs) New functionalized EbNPsBroad spectrum antimicrobial/ Yes Yes antisporal/ antiviral agentEfficiency ++ +++ (up to 10X higher efficiency) Silver-content (prioritypollutant) High Low (reduction factor up to 10) Matrix Persistent(metallic Ag based) Degradable (biopolymer-based) Suitability forcustomized Low (one function) High (functionalization with functionalityother active agents possible) Regulatory Concerns Yes (under scrutiny byUS EPA) Minimized (collaboration with EPA) Comparative activity towardsMedium (tested at US EPA) Low (tested at US EPA) mammalian cellsBioactivity post-utilization Yes (metallic core remains active)Minimized (depleted of active agent) Scalability Difficult Yes(continuous flow production (mostly batch production) possible)Comparative concentration [wt %] Low High (10 to 100 higher) in solutionPrice $400/g AgNPs in solution $4.0/g EbNPs in solution (estimate,PlasmaChem GmbH, (conservative estimate) 2012)

As mentioned above, the qualitative antimicrobial test on P. aeruginosacan distinguish the antimicrobial effect of PDADMAC from the effect ofsilver (FIG. 27). BPEI AgNPs and AgNO₃ solutions exhibited no completeantimicrobial effect after 30 minutes incubation time. The controlsample IAT EbNP and the IAT EbNPs coated with PDADMAC also did notresult in any significant effect. Thus, the sample Ag-EbNPs-PDADMAC wasthe only one that exhibited complete or 100% antimicrobial efficiencyafter 30 minutes. The supernatant of the sample Ag-EbNPs-PDADMAC did notshow any antimicrobial effect after 30 minutes of incubation time. Theresults suggest that the antimicrobial action of Ag-EbNPs-PDADMAC isdelivered by silver ions. Comparing all active agents tested in terms ofantimicrobial efficiency, we establish that Ag-EbNPs-PDADMAC againproved most effective.

presents a comparison of the properties of common AgNPs with our novelsilver-infused EbNPs.

Hypothesis of the Antibacterial Mechanism of Ag-EbNPs-PDADMAC.

The antibacterial effect of Ag-EbNPs-PDADMAC, is a combinatorial effectof the antimicrobial properties of Ag⁺ ions and of the quarternizedamine PDADMAC. For the bacteria not susceptible for bactericidal aminessuch as P. aeruginosa, ^(38, 39) the antimicrobial effect is based onthe bactericidal activity of silver-ions. We suggest that the Ag⁺ ionsare weakly bound to the EbNP binding sites and locally concentrated onthe EbNP surface. These Ag⁺ ions may be surface active and could bereleased upon contact with a bacteria cell membrane. A possiblemechanism of the antimicrobial Ag-EbNPs-PDADMAC activity is illustratedin FIG. 28. Ag-EbNPs-PDADMAC particles are electrostatically attractedto the negatively charged bacteria cell membrane, and will eventuallyadhere to it. As the control sample of EbNPs with PDADMAC but withoutAg⁺ ions did not show antimicrobial effects towards P. aeruginosa, wesuggest that the particles by themselves may not destroy the integrityof the cell membrane, which could result in cell lyses and thereforecell death. Hence, we suggest that the antimicrobial effect stemspredominately from Ag⁺ ions, which may be released by the EbNP systemtowards the bacteria. As the Ag⁺ ions may eventually migrate into thecell, they could adversely affect bacteria cell functions and therefore,lead to cell death. Conclusions. We developed a new class ofmicrobicidal nanoparticles with increased efficiency and improvedpost-utilization safety. In contrast to AgNPs, the Ag-EbNPs-PDADMACsystem is synthesized via green chemistry and employs degradable, benignand sustainable materials. Since these EbNPs can promote significantlyhigher antimicrobial activities in terms of Ag equivalents in comparisonto persistent AgNPs, their environmental footprint is largely reduced.Furthermore, antimicrobial EbNPs are benign towards mammalian cells incomparison to AgNPs at equivalent silver concentration. As the EbNPtechnology is flexible and may be applied to a wide range of activeagents, functionalized EbNPs may be suitable to substitute a wide rangeof applied metal nanoparticles.

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Additional data, examples and embodiments may be found in Appendix Aattached to the specification of U.S. provisional patent application No.61/776,274, filed Mar. 11, 2014, the benefit of which application isclaimed by the present application, and the contents of whichapplication are incorporated by reference herein in its entirety.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object(s) of the article.By way of example, “an element” means one or more elements.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. The present inventionmay suitably “comprise”, “consist of”, or “consist essentially of”, thesteps, elements, and/or reagents described in the claims.

The following Examples further illustrate the invention and are notintended to limit the scope of the invention.

It is to be understood that, while the invention has been described inconjunction with the detailed description, thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below. All publications,patents, and patent applications cited in this specification are hereinincorporated by reference as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A cytotoxic particle comprising: (a) a discrete,solid, nanoparticle core wherein said nanoparticle core is comprised ofa plant derived lignin; (b) a cytotoxic metal ion reversibly bound tothe discrete nanoparticle core; and (c) a bioadhesive adsorption layercoating the discrete nanoparticle core and the metal ion, wherein saidbioadhesive adsorption layer comprises a cationic polymer.
 2. Thecytotoxic particle of claim 1 wherein the reversibly bound metal ion isAg⁺, Ag²⁺, Ag³⁺, Co²⁺, Cu¹⁺Fe³⁺Cu²⁺, Fe²⁺, Ni²⁺, or Zn²⁺.
 3. Thecytotoxic particle of claim 1 wherein the cationic polymer is apolyamino polymer.
 4. The cytotoxic particle of claim 3, wherein thecationic polymer is a polyamino polymer selected from the groupconsisting of: branched polyethyleneimine (BPEI), polyallylaminehydrochloride (PAH), polydiallyldimethylammonium chloride (PDADMAC),polyethoxylated tallow amine (POEA), polyethyleneimine (PEI), andpolylysine.
 5. The cytotoxic particle of claim 1, wherein the cationicpolymer comprises primary, secondary, tertiary, or quaternized aminefunctional group.
 6. A coated article comprising a surface wherein atleast a portion of the surface is coated with the cytotoxic particle ofclaim
 1. 7. The coated article of claim 6, wherein the coated article isan air filter, an article of clothing, an article of hygiene, a buildingmaterial, a face mask, a food stuff package, a medical device, or aseed.
 8. The coated article of claim 6, wherein the coated article is abandage, a biological implant, a dressing, a medical scaffold, asurgical instrument, or a wound covering.
 9. A cytotoxic particlecomprising: (a) a discrete nanoparticle core wherein said nanoparticlecore comprises a biodegradable lignin; (b) a cationic and cytotoxicmetal ion reversibly bound to the discrete nanoparticle core; and (c) asingle layer of polyamino cationic polymer coating the discretenanoparticle core and the cytotoxic metal ion.